In vivo examination of pathogenicity and virulence in environmentally isolated Vibrio vulnificus

Abstract Human exposure to Vibrio vulnificus, a gram‐negative, halophilic environmental pathogen, is increasing. Despite this, the mechanisms of its pathogenicity and virulence remain largely unknown. Each year, hundreds of infections related to V. vulnificus occur, leading to hospitalization in 92% of cases and a mortality rate of 35%. The infection is severe, typically contracted through the consumption of contaminated food or exposure of an open wound to contaminated water. This can result in necrotizing fasciitis and the need for amputation of the infected tissue. Although several genes (rtxA1, vvpE, and vvhA) have been implicated in the pathogenicity of this organism, a defined mechanism has not been discovered. In this study, we examine environmentally isolated V. vulnificus strains using a zebrafish model (Danio rerio) to investigate their virulence capabilities. We found significant variation in virulence between individual strains. The commonly used marker gene of disease‐causing strains, vcgC, did not accurately predict the more virulent strains. Notably, the least virulent strain in the study, V. vulnificus Sept WR1‐BW6, which tested positive for vcgC, vvhA, and rtxA1, did not cause severe disease in the fish and was the only strain that did not result in any mortality. Our study demonstrates that virulence varies greatly among different environmental strains and cannot be accurately predicted based solely on genotype.


| INTRODUCTION
Vibrio vulnificus is a gram-negative, halophilic bacterial species that is found in estuarine and coastal waters.This naturally occurring environmental human pathogen is a great concern for public health due to its routine isolation from the water column, sediment, and shellfish.There are roughly 80,000 cases of Vulnificus sp.infections, or vibriosis, each year in the United States.Although the number of infections caused by V. vulnificus is a relatively low percentage of total vibriosis cases, the severity of injection makes V. vulnificus a formidable threat (Scallan et al., 2011).Most V. vulnificus cases originate from wound infections and septicemia, with only 5% involving gastroenteritis (Scallan et al., 2011).Hospitalization rates for V. vulnificus infection are high, in 92% of cases, and the mortality rate is 35% (Scallan et al., 2011).If infection occurs through an open wound, V. vulnificus infection can lead to sepsis and necrotizing fasciitis.The mortality rate of V. vulnificus when it invades the bloodstream increases to 60%.In seafood-related deaths in the United States, V. vulnificus is the major cause, responsible for 95% of these infections.Despite the severity and concern with V. vulnificus infections, it is still poorly understood which genes are directly involved in pathogenicity (Al-Assafi et al., 2014;Linkous & Oliver, 1999).
Understanding the virulence of V. vulnificus has been underway for several years, yet little is known about the pathogenicity mechanism or which specific genes are involved in virulence for this species.Several strains can produce cytotoxic effects in human epithelial cell lines, but research has heavily focused on the strains from clinically isolated sources (Hiyoshi et al., 2010;Raimondi et al., 2000).Little work has been done to test the virulence capabilities of environmentally derived strains, based on the premise that few strains that naturally persist in the environment cause serious disease, or occur at low, infrequent incidences (Baker-Austin et al., 2008;DePaola & Kaysner, 2004).However, when environmentally derived strains of V. vulnificus and Vibrio parahaemolyticus were compared to clinical strains using a human gastrointestinal epithelial cell line, both clinically and environmentally isolated strains caused similar degrees of damage to human cells in vitro (Klein, 2018).
There have been implicated virulence genes proposed to contribute to the pathogenicity process of the organism, but unlike its relative Vibrio cholerae, there is still no defined pathogenicity mechanism.V. vulnificus is commonly broken down into groups based on biotypes based on their biological and biochemical properties: biogroup 1, biogroup 2, and biogroup 3 (Linkous & Oliver, 1999;Oliver, 1989).Biogroup 1 strains are derived most frequently in the clinic, from patients who are actively suffering from a V. vulnificus infection.Biotype 2 is an extremely rare human pathogen isolated from diseased eels (Amaro & Biosca, 1996).A more recent addition to the clade of V. vulnificus is Biotype 3, which has thus far only been isolated in Israel (Efimov et al., 2013).The genome of Biotype 3 is more closely related to the Biotype 1 genomes, with 90% similarity, whereas the Biotype 2 genome has 87% similarity.Previous studies have suggested that pathogenesis occurs through the penetration of cells by extracellular proteins released by the invading bacteria, which causes tissue damage, especially to tissue of vascular nature (Al-Assafi et al., 2014;Jeong & Satchell, 2012).
V. vulnificus is also commonly broken down into two genotypes.
These two groups are established by the presence of a specific virulence-correlated gene (vcg), either type E or type C. Biotype 1 strain contains the vcgC gene (Rosche et al., 2005).The vast majority (90%) of clinically isolated strains of V. vulnificus contain the vcgC variant, while the vast majority of environmentally isolated strains contain vcgE (87%) (Rosche et al., 2005).Despite this, the vcg gene likely does not contribute to virulence as it does not code for a protein.Furthermore, many environmental isolates of V. vulnificus also contain the vcgC variant, suggesting that the presence of vcg may not be an accurate way of predicting virulence in the environmental setting (Klein, 2018).
Although no mechanism has been defined, several genes have been identified as possible virulence factors in V. vulnificus.The hemolysins (vvhA and vvhB), toxins (rtxA1), siderophores, outer membrane proteins and lipopolysaccharides (LPS), and flagella components have also been implicated in V. vulnificus virulence (Goo et al., 2006;Jeong & Satchell, 2012;Jones & Oliver, 2009;Kim et al., 2010Kim et al., , 2012;;Yokochi et al., 2013).The gene vvhA encodes a hemolysin that induces cytolysis and death by making pores in erythrocyte cell membranes (Kim et al., 2010).Hemorrhagic damage and skin necrosis are triggered by the protease vvpE.This results in vascular permeability, causes edema, and is ultimately lethal in mouse models of infection (Kothary & Kreger, 1987).The VvpE gene has also been implicated in the invasiveness of V. vulnificus.The protein encoded by VvpE may induce proteolytic cleavage of lactoferrin and IgA antibodies (Kim et al., 2007).The rtxA1 gene is one of the most characterized virulence factors of V. vulnificus (Kim et al., 2016).This gene is a cytotoxin with multiple functions, triggering rearrangement of the cytoskeleton, hemolysis, cytotoxicity, and facilitating tissue invasion, which can ultimately lead to lethality in infected mice.
Despite this work, the exact mechanism of V. vulnificus virulence remains unresolved.
To address variation in the virulence of environmental isolates of V. vulnificus, we utilized a Zebrafish model.Zebrafish (Danio rerio) have successfully been used to study the virulence and pathogenic capabilities of many different types of bacteria, including several Vibrio species (Bergeron et al., 2017;Mitchell & Withey, 2018;Nag et al., 2020;Neely et al., 2002;Paranjpye et al., 2013).Although Zebrafish have a lower internal body temperature than mammalian model systems, which may result in slower bacterial growth, the Zebrafish model has been shown to fully recapitulate the full life cycle of bacterial infection (Mitchell & Withey, 2018).Zebrafish have innate and adaptive immune systems, so the symptomatic effects of injection are nearly identical to humans (Da'as et al., 2011;Stemple & Driever, 1996;Sullivan & Kim, 2008).Here, we utilized the Zebrafish model to understand how the virulence of environmentally isolated V. vulnificus strains compares to clinically isolated strains.We discovered a range in virulence, which differs greatly between strains, and our data suggest that the use of vcgC and vcgE as potential predictors of genotype and virulence may not be a reliable tool.

| Strain isolation
Environmental V. vulnificus strains were isolated from lower salinity waters in Winyah Bay and the Waccamaw River near Georgetown, SC, USA (33°20′ N, 79°12′ W) by Daniel Tufford.CHROMagar Vibrio (DRG International) was used for the initial plating of the water samples to isolate V. vulnificus strains (following the US Food and Drug Administration protocol) (DePaola & Kaysner, 2004).After this initial isolation, V. vulnificus strains were routinely cultivated on saline lysogeny agar (SLA; per L; 10 g tryptone, 5 g yeast extract, 15 g NaCl, 15 g Bacto Agar) and TCBS agar (Thiosulfate Citrate Bile Salts Sucrose Agar), a selective and differential growth medium specific to Vibrio spp., which is recommended to use for the selective isolation and cultivation of Vibrio spp.from clinical specimens (Hardy Diagnostics).V. vulnificus strain JY1701 was isolated environmentally via methods described by Rosche et al. (2005).

| Whole genome sequencing
Genomic DNA was isolated using a DNA purification kit following the manufacturer's protocol for Gram-negative organisms (Wizard Genomic DNA Purification kit, Promega).After extraction, the DNA was quantified via Quibit fluorimetry and libraries were prepared.V.
vulnificus Aug-WR2-BW was sequenced using an Illumina MiSeq (V3 26300 base) at the Indiana University Center for Genomic Studies.

| Zebrafish husbandry and care
Zebrafish (D. rerio) used in this study were from the Tübingen strain.
Maintenance and breeding were conducted in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines, which included housing zebrafish in reverse osmosis-filtered water-flow tanks maintained at 28.5°C.
Zebrafish were fed a commercial diet (Skretting) once per day.The salinity of zebrafish water was maintained at 0.5 g/L.Conditions used for virulence studies are described below.

| Bacterial growth conditions
Table 1 contains the strains of bacteria used in this study.Overnight cultures of strains were grown in a medium containing 15 ppt NaCL SLA broth using a shaking incubator at 37°C.Two PBS washes were conducted on cultures before inoculation.We performed serial 10-fold dilutions of the cultures before plating on 15 ppt NaCl SLA to confirm the concentration of the inoculum.Controls for this experiment included PBS buffer as well as a non-virulent strain of Vibrio, Vibrio pacinii DSM 19139 T .V. pacinii was grown overnight in 15 ppt NaCl SLA broth at 23°C.

| PCR virulence gene screening
After overnight incubation at 37°C in SLB, isolates were centrifuged and transferred to sterilized distilled water, where DNA was extracted through boiling at 95-100°C for 20 min.For all PCR reactions, 1 μL of the sample was used in each reaction.The species of the bacteria was confirmed by amplification of the recombinase A (recA) gene (Thompson et al., 2005).A common housekeeping gene that is typically used for species identification is the 16s rRNA gene; however, in vibrios, this gene is too highly conserved and does not allow for species resolution (Thompson et al., 2004).Amplicons were sent off for sequencing (Eurofins) and a phylogenetic tree using comparison sequences from NCBI was created.The protocol and primers that were used followed the protocols outlined by Thompson (2005).PCR products for recA (790 bp) were separated on a 1.5% agarose gel.The ABI Prism 3730 DNA analyzer was used for sequencing.Once sequences were received, they were edited, and the Kimura 2 parameter model with Mega 7 was used to make the T A B L E 1 Gene distribution of Vibrio vulnificus strains used in zebrafish inoculations.
Note: Bold indicates strains from a clinical source.Superscript T indicates type strain.
PIPES ET AL.
V. vulnificus strains were screened for the following virulence factors: vvhA, vcgC, vcgE, vvpE, and rtxA1.The virulence correlated genes (vcgC and vcgE) variants were used as indicator genes to differentiate between strains thought as avirulent (vcgE positive) and pathogenic (vcgC positive).PCR primers of Warner and Oliver (2008b) were used to amplify vvhA (410 bp), vcgE (199 bp), and vcgC (97 bp).The primers of Liu et al. (2007) were used to amplify the rtxA1 gene and the primers of Jeong et al. (2001) were for the vvpE (697 bp) gene.PCR products for all virulence genes were separated on a 1.5% agarose gel.

| Intraperitoneal challenge
For challenge experiments, zebrafish were anaesthetized in 25 mg/L tricaine.Once fully anaesthetized, zebrafish were removed from the anesthesia tank and placed on a 25 mg/L tricaine-soaked sponge for the duration of the injection procedure.Fish were challenged using an intraperitoneal (IP) injection of 10 μl of the inoculum using a 33gauge needle on a Hamilton syringe (Hamilton).The needle was placed at the midway point located between the pectoral fin and the anus in accordance with previously published protocols (Lefebvre et al., 2009;Paranjpye et al., 2013).After injection, Zebrafish were housed in off-system individual glass aquariums, with water supplied from the system water of the zebrafish facility.Water temperature was maintained at room temperature or slightly above (26-28°C).A lethal dose of tricaine was used to sacrifice any surviving fish at the experimental endpoint of 3 days.All aquariums and water were disinfected with 10% bleach solution.

| Virulence evaluation and statistical analysis
Each strain was tested on three fish per trial, and each trial was repeated two times.Fish were monitored for 7 h post initial IP injection.Fish were housed for 3 days with monitoring, for several hours each consecutive day.Fish were viewed grossly to determine any signs of external injury, such as swelling, redness, and lesions, an increase in fecal production, changes in swimming patterns, changes in breathing patterns, and death.To determine how the bacteria infiltrate the fish body, some fish were sectioned into three regions: head, abdomen, and tail regions.These sections were weighed, homogenized, and then resuspended into 900 microliters of PBS.
These were then serially diluted 10-fold and were plated onto TCBS agar to re-isolate the Vibrio bacteria to get colony counts and determine the concentration of Vibrio cells per gram of fish tissue per region.A one-way analysis of variance (ANOVA) using SPSS was performed comparing the means of fish lethality for each clade of tested Vibrio strains: vcgC-positive, vcgE-positive, and both vcgC and vcgE-positive.The significance level for the test was set at a p-value of 0.05.A one-way ANOVA was also used to compare the means of fish lethality between the clinically isolated strains and the environmentally isolated strains.A significance level of 0.05 was also used.

| Genome gazing
V. vulnificus WR2-BW, JY1701, and 27562 T genomes were used to genome gaze and compare the differences in gene makeup between the three strains.The RAST SEED Viewer application was used to compare the genomes of the three strains, which were compared using a sequence-based function (Aziz et al., 2008;Overbeek et al., 2005Overbeek et al., , 2014)).The comparison tables were downloaded and using the most virulent strain from the three of them as the reference organism, gene profiles of missing genes from the two less virulent strains were categorized.

| RESULTS
Several environmental V. vulnificus strains were isolated from the lower salinity waters of the Waccamaw River and the Winyah Bay areas near Georgetown, SC, USA.Gene sequences of the recA gene were used to confirm that the environmentally isolated bacterial species were V. vulnificus, based on the phylogeny and percent similarity identity scores compared to reference genes of the recA gene in confirmed and sequenced strains of V. vulnificus from NCBI GenBank (Figure 1).For all species confirmed to be V. vulnificus used in the study, 100% tested positive for containing the vvhA gene.Of the 13 environmentally isolated strains used, seven strains (54%) were vcgE variant positive, two strains (15%) were vcgC variant positive, and four strains (30%) contained both variants of the vcg gene.For the vgcE-positive strains, all but one contained the rtxA1 gene, and five of the seven were positive for vvpE.Strain Sept WR1-BW4 was the only strain of the vgcE clade that was negative for rtxA1 and it was also negative for vvpE.The other strain negative for vvpE was Oct 05-25-BW.Looking at the vcgC clade, strain Oct SF 05-20-BW tested positive for both rtxA1 and vvpE, whereas strain Aug WR1-BW6 tested positive for only rtxA1.All strains that contained both vcg variants also contained rtxA1 and vvpE (Table 1).
To examine virulence, three fish per strain were injected into the abdomen with 10 µL of bacterial culture with a cell concentration of 10 3 CFUs/mL.Virulence varied greatly between individual strains (Figure 2).The most common early symptoms in the fish included diarrhea and site injection redness and irritation.As the infection progressed, many fish displayed difficulty swimming and maintaining positive buoyancy, signs of labored breathing, and many became lethargic, lying prone on the bottom of the aquarium.In the more virulent strains, there was enterohemorrhagic activity, and upon dissection, many fish that succumbed early in the infection had a lower blood volume and visible tissue damage when compared to fish that survived the entire trial.A total of 102 fish were used in this study (17 strains, two trials for each strain, each trial involving three inoculated fish).Out of the total study, 67 (65%) fish succumbed as a direct result of the V. vulnificus infection (Table 2).For the environmentally isolated strains, 58% of the fish directly succumbed to the V. vulnificus infection (Table 2).For the clinically isolated strains, 91% of the inoculated fish succumbed to the bacterial infection.The clinically isolated strain ATCC BAA-86 was the most virulent of all tested strains, where both trials with this organism resulted in 100% mortality within 24 h of exposure.Comparatively, the least virulent strain was V. vulnificus strain Aug WR1-BW6, which was not lethal to any fish tested in the two trials, and the fish produce a difference in mean lethality during the trials.This analysis showed no statistically significant difference in the means between the three groups, with a p-value of 0.687.The one-way ANOVA looking at the means of lethality between the clinically isolated strains and the environmentally isolated strains also showed no significant difference between the two, with a p-value of 0.051.
Fish that were injected with strains BAA-86 and 05-21-BW1 were used to determine cell recovery per gram of tissue in three sections of the fish: head, abdomen, and tail.These fish regions were homogenized and serially diluted onto TCBS agar.For BAA-86, the results showed that the highest recovery of bacteria came from the abdomen region, with the average of six fish being 1.58 × 10 9 CFUs g −1 , the tail region with the next highest concentration at 1.07 × 10 8 CFUs g −1 , and the head region with the lowest at 2.35 × 10 7 CFUs g −1 .The results for environmental strain 05-21-BW1 held consistent when compared to BAA-86 with the abdomen region with the highest cells recovered per gram of tissue at 1.55 × 10 9 CFUs g −1 , but for the environmental strain, the head had the next highest cells recovered at 5.65 × 10 7 CFUs g −1 , and the tail region with the lowest at 8.18 × 106 CFUs g −1 .
Of the three strains with full genome sequences (Accession numbers for V. vulnificus JY1701, V. vulnificus ATCC 27562 T , and V. vulnificus WR2-BW, respectively: AFSY00000000, NZ_AMQV00000000, GCA_003798485.1),V. vulnificus JY1701 had the highest level of virulence, with 100% lethality.Both strains ATCC 27562 T and WR2-BW resulted in 67% lethality.JY1701 and WR2-BW are vcgE-positive strains, whereas ATCC 27562 T is a vcgC-positive strain (Table 1).xJY1701 was set as the reference genome for comparative genome analysis across the strain due to the high virulence of this strain.There were a total of 305 unique genes present in JY1701 that were not present in either WR2-BW or ATCC 27562 T .Only 47 of those 305 genes have a known function (Table 3).The rest of the unique genes (258 genes, or 84.5% of the total unique genes) were hypothetical proteins, genes of unknown function, or phage-related genes.Four candidate virulence-factorassociated genes were identified in V. vulnificus JY1701 that were not present in WR2-BW and ATCC 27562 T .They are T1SS-secreted agglutinin RTX, a virulence-associated E gene, a putative integrase, and a WzxE protein.These genes may represent previously unknown virulence factors in V. vulnificus.

| DISCUSSION
The pathogenicity of V. vulnificus is complex, undefined, and varied across strains.Here, we show a complicated mechanism for V.
vulnificus virulence, where virulence between those isolated from the environment differs greatly and does not necessarily correlate with the presence of previously implicated virulence-related genes: vvhA and vvhB, rtxA1, and vvpE.When examined in our system, the clinically isolated vcgC-positive strains were highly virulent in the zebrafish.Yet, the presence of vcgC in our study did not always reflect the highest virulence capabilities.The least virulent strains from the study were V. vulnificus Sept WR1-BW6, June 05-25-SW1, and Sept 05-20-BW4, all of which contain a copy of the vcgC gene.
Our study included seven strains lacking vcgC and positive for vcgE alone.Of those, six of the seven strains were as virulent as the least virulent clinically isolated strain of V. vulnificus (ATCC 27562 T ), where each of these strains resulted in 67% mortality.
Previous studies have shown that V. vulnificus isolates from oysters show an overwhelming proportion of vcgE-positive strains, and this has been considered a reason why the incidence of V.
vulnificus infections is relatively low; if vcgC strains are not predominant, then it is less likely for a person to consume or encounter "virulent strains" (Warner & Oliver, 2008a).There may be an important ecological reason as to why the vcgE genotype does predominate in oysters.However, this genotyping protocol implies that the vcgE strains are incapable of causing disease.Our study demonstrates that there are virulent vcgE strains.In addition, cytotoxicity trials in previous studies have shown that vcgE strains are capable of destroying epithelial cells and causing disease (Klein, 2018).The introduction of live bacteria into zebrafish is not a trigger of death as a process, as not a single fish that was inoculated with Vibrio pacinii DSM 19139 T , an avirulent bacterial species, died or showed any signs of illness or distress.Relying on genotype alone (vcgC vs. vcgE) as a marker for pathogenic versus nonpathogenic status may not be reliable.
Through genome gazing of published data sets from tested strains of V. vulnificus, it was determined that 305 genes were unique to V. vulnificus JY1701 when compared to V. vulnificus ATCC 27562 T and Aug WR2-BW.Of those 305 genes, only 47 have a known or defined function.The remaining genes are hypothetical, undefined, or phage-related.We identified four genes that may contribute to virulence-related function.Those genes include the T1SS-secreted agglutinin RTX, wzxE, virulenceassociated E, and a putative integrase.
The Type 1 secretion systems (T1SS) contain genes present in many gram-negative bacteria.These genes form a secretion and delivery mechanism for several different virulence factors, including hemophores, proteases, and lipases to target cells (Masi & Wandersman, 2010).The rtxA1 toxin is known to be associated with Type 1 secretion models (Lee et al., 2008).Within all three genomes examined, multiple genes related to the T1SS secreted agglutinin RTX; however, WR2-BW and ATCC 27562 T only contained two copies of the three genes that made up the system and did not contain rtxA1.One hypothesis for reduced virulence may be a reduction in the delivery of the RTX toxin into the host organism by strains lacking this gene compared to JY1701.
The wzxE protein is another interesting candidate for virulence.
The less virulent V. vulnificus Aug WR2-BW contains a similar wzx gene within a PAI (Klein et al., 2018); however, it is a variant of the wzxE gene encoding the O-antigen flippase, rather than wzxE.This family of genes is considered to be a virulence-associated factor, as host cell damage is not directly caused, but it does contribute to  however, the wzx/wzy pathway remains partially undefined and research is still needed to fully understand this pathway and all its functions (Hong et al., 2017).
The virulence-associated E proteins belong to the family of proteins that contain a p-loop motif, or phosphate-binding loops.
Virulence-associated proteins have been identified in other microorganisms, including Streptococcus and Rhodococcus species (Ji et al., 2016;Okoko et al., 2015).Mice that were exposed to a strain of Streptococcus suis serotype 2 that had a functional copy of the virulence-associated E protein (vapE) exhibited more severe symptoms, which included behavioral symptoms of apathy and depression, along with anorexia, fever, emaciation, and neural disorders.These mice succumbed to infection within 2 days.Mice exposed to a vapE mutant exhibited less severe clinical symptoms and all recovered within a week (Ji et al., 2016).The role that vapE plays in pathogenicity is poorly understood, but these trials indicate a role for vapE in the virulence of this species of Streptococcus.Because of vapE's undefined overall function and role in pathogenicity mechanisms, it is unclear how its role in V. vulnificus virulence is involved, but strong evidence from here and others suggests it may play a role.
The putative integrase may be involved in virulence differently, as a site of recombination and integration of foreign DNA (Hacker & Carniel, 2001;Hacker & Kaper, 2000).Vibrio cholerae, a close relative of V. vulnificus, also has putative integrase genes, the accessory colonization factor (ACF), and the toxin-coregulated pilus (TCP) (Kovach et al., 1996).ACF and TCP are involved in the colonization of host cells and greatly aid in the ability to successfully colonize the host intestinal epithelial tissue (Kovach et al., 1996).Oliver, 1983).In many cases, blood serum iron is unavailable to microorganisms due to inhibitory effects, which creates an iron deprivation for microorganisms while in the bloodstream (Weinberg et al., 1978).When iron-containing compounds that are more biologically available have been injected directly into the blood during animal infection models, there has been an increase in microbial numbers (Holbein et al., 1981;Kochan et al., 1977).Thus, the ability of V. vulnificus to produce siderophores may provide an important but under-recognized virulence mechanism to provide the high levels of iron required for sustained survival.Additionally, V.
vulnificus can use exogenous siderophores produced by other bacterial species (Alice et al., 2008;Aso et al., 2002;Barnes et al., 2020).Due to the nature of the IP injection we used in this study, there is a possibility that some natural skin microbiota of the zebrafish were introduced along with the Vibrio bacteria during injection.These include Enterobacteriaceae sp, Flavobacterium sp, and Sphingobacteriales sp, among others, which are also known to produce siderophores (Nakatani & Hori, 2021).Although the skin bioburden would be significantly less than the introduced V. vulnificus dose, these strains may also play a role in iron acquisition by V.
vulnificus.Consequently, iron acquisition in V. vulnificus may play an important role in sustaining an infection.Continued examination into the siderophore usage between the virulent strains versus less virulent strains may provide further evidence for the importance of this mechanism.
Here, we have identified and characterized several environmental isolates of V. vulnificus with a range of virulence in vivo.Our work demonstrates the importance of understanding that virulence between different environmental strains varies greatly and cannot accurately be predicted based on genotype alone.The pathogenicity mechanism for this organism remains elusive and hidden in the genome, requiring full genome sequencing of more environmental strains to aptly determine which genes may be involved in virulence that remain to be identified.
exhibited very little symptomatic response to the bacterial injection.Inoculation with Vibrio pacinii DSM 19139 T , an avirulent bacterial species, and PBS alone were used as controls for injections.No lethality, illness, or distress was observed in these treatment groups.A one-way ANOVA statistical test was completed to compare the means of lethality between the three clades of tested strains: vcgE-positive, vcgC-positive, and both variant positive, based on the hypothesis that the means between the different gene clades would F I G U R E 1 Maximum-likelihood phylogeny of recA gene sequences (Kimura 2-parameter model).The bootstrap values represent 1000 replications.NCBI GenBank was the source for the acquisition of the reference sequences.Bolded sequences are the environmental strains used in this study.
Lethality rate of fish totaled from two independent trials of Vibrio vulnificus strains.Clinically or independently isolated strains (a) include ATCC-27562 T , ATCC-BAA-86, ATCC 33817, and JY1701.All other strains are environmentally isolated for this study (b).Controls for the experiment included fish injected with PBS buffer and an avirulent Vibrio species, Vibrio pacinii.Black bars indicate vcgC-positive strains.Light gray bars indicate vgcE-positive strains.Dark gray bars indicate strains positive for both vcgC and vcgE.
pathogenesis and aids in infection.A related gene, belonging to the oligosaccharide flippase family based on the NCBI Protein Blast score (99%), was found in JY1701.The most prominent component of the outer membrane of Gram-negative bacteria, lipopolysaccharide (LPS), is known to induce fevers (Jones & Oliver, 2009; McPherson T A B L E 2 Fish lethality rate of Vibrio vulnificus strains used in zebrafish inoculations.
Note: Totals come from two independent trials of each strain.Each trial was repeated twice.Bold indicates strains from a clinical source.Superscript T indicates type strain.Results from genome gazing at the genes present and unique to strain Vibrio vulnificus JY1701 and not present in strains V. vulnificus 27562 T or WR2-BW.
et al., 1991).Genes that fall under this category of translocation of lipid-linked oligosaccharides are used in activities, such as cell wall construction, polysaccharide synthesis, and protein glycosylation; The genes in this table are genes with known functions.Hypothetical proteins, genes with unknown function, or phage-related genes are not included.Bolded genes indicate genes that may have a possible virulence-related function.